ABSTRACT Microglia,-the resident macrophages of the central nervous system (CNS), are thought to be important contributors to CNS health and' disease but their exact functional roles are still mysterious. Direct CNS damage, as well as peripheral injuries and addictive drug exposure, all induce microglial activation. A longstanding limitation in investigating microglia is the lack of good tools to distinguish and manipulate microglia specifically without altering macrophages or other immune cell types. In this proposal we will use new tools that our lab has recently generated to specifically identify and genetically manipulate microglia in order to investigate their function. First we will use these tools to develop the first in vitro culture system in which purified microglial cells can be cultured in defined serum-free medium with high survival in a non- activated state. This will enable us to directly test their specific functions for the first time including their ability to promote neuronal and glial survival, or to regulate synapse formation and function, as well as to elucidate the specific signals that induce their activation and to assess how their normal functions are altered when microglia become activated. Second, we will perform metabolomics analysis to investigate nature of the chemical signaling molecules that resting and activated microglial cells secrete. Third, we will use the new genetic tools we have developed to ablate microglia to elucidate their role in the establishment and maintenance of reactive gliosis and neuropathic pain. Lastly vye will identify the specific molecular mechanisms through which microglial cells influence these processes. Presently, drugs available for treatment of pain, such as opiates, suffer from undesired side effects and the development of tolerance. The purpose of the proposed research is to determine the molecular events that produce glial cell mediated neural circuitry modifications, opioid tolerance and addiction, as well as neuropathic pain in order to identify novel therapeutic targets. The findings will lay the groundwork for future research in understanding the role of microglia in drug- induced plasticity in reward-related neuronal circuits.